The goal of this proposal is to explore the regulation and contribution of the enzyme xanthine oxidoreductase (XOR) in the pathogenesis of ventilator-associated lung injury (VALI). XOR plays an essential role in organ dysfunction, including acute lung injury (ALI), because of its ability to generate reactive oxygen species (ROS). Using both in vitro and in vivo systems, our laboratory has demonstrated transcriptional up-regulation of this enzyme by hypoxia, endotoxin, and cytokines (all known protagonists in the development of ALI) as well as post-translational modification and activation of XOR after phosphorylation by the Mitogen Activated Protein Kinase (MAPK) pathway. Furthermore, we have recently linked VALI-associated capillary vascular leakage to p38 MAPK/XOR signaling. Oxidative stress, which is a central component of alveolar cell injury and capillary leakage in this model, can be prevented by XOR inhibition. In preliminary experiments that form the basis of this application renewal, we now demonstrate that mechanical stress, in the context of high tidal volume ventilation, leads to profound re-arrangement of the pulmonary endothelial cytoskeleton, apoptosis, and capillary leakage, all of which can be prevented by inhibition of the p38 MAPK/XOR signaling pathway. We hypothesize that p38 MAPK/XOR activation in response to mechanical stress is a nodal event in oxidative stress, endothelial cytoskeletal remodeling, apoptosis, and endothelial barrier dysfunction. In this application, we will utilize both in vitro and in vivo models to determine the role of mechanical stress mediated p38 MAPK/XOR activation in VALI. We will also delineate the role of XOR-derived ROS in the activation of specific components of the apoptotic cascade, cytoskeletal remodeling, and the mechanisms leading to capillary leakage. Specific Aim 1 will define the mechanisms and sites of XOR post-translational modification by the MAP kinase pathway. Specific Aim 2 will determine the role of XOR and mechanisms involved in endothelial cell apoptosis in response to mechanical stress. Specific Aim 3 will define the contribution of XOR in mechanical stress mediated cytoskeletal rearrangement and capillary leakage. We anticipate that the studies proposed in this application will identify key molecular targets for future therapeutic strategies aimed at decreasing mortality in VALI. PUBLIC HEALTH RELEVANCE: Mechanical ventilation (MV) is the cornerstone of treatment for patients with acute lung injury, a disease which carries great morbidity and mortality thus representing a significant public health problem. Since MV can cause mechanical stress through over-distension of the alveolar spaces, furthering lung injury and causing ventilator- associated lung injury or VALI, we propose to investigate the molecular mechanisms leading to the pulmonary capillary leakage (edema). We will use genetically manipulated mice and pharmacological inhibitors to test our hypotheses that the enzyme xanthine oxidoreductase (XOR) and the p38 MAP kinase signaling pathway play crucial roles in the pathogenesis of VALI, and anticipate that the studies proposed will identify key molecular targets for the generation of therapeutic strategies aimed at decreasing the mortality of VALI.